PROJECT SUMMARY/ABSTRACT Chronic wounds affect approximately 6.5 million patients in the United States and represent $25 billion in annual health care costs. This burden is rapidly increasing due to escalating health care costs, an aging population, and the rising incidence of diabetes and obesity. Studies demonstrate that biofilms contribute to wound chronicity, and that Staphylococcus aureus is a prevalent species found in chronic wound biofilms; however, how biofilms contribute to the delayed re-epithelialization remains unclear. It was recently demonstrated that planktonic S. aureus and S. aureus biofilms have differential lethal effects on keratinocytes (KC). Therefore, it is hypothesized that planktonic S. aureus produce soluble factors that induce KC necrosis; whereas S. aureus biofilms produce soluble factors that mediate specific apoptotic pathways in KC. This hypothesis will be tested by comprehensively investigating KC cultures exposed to either biofilm-conditioned medium or planktonic-conditioned medium for apoptotic characteristics, including phosphatidylserine translocation and DNA fragmentation. Next, KC cultures will also be analyzed for the induction of the intrinsic and/or extrinsic apoptotic pathways. More specific molecular analysis of KC apoptosis will be investigated using a large format quantitative polymerase chain reaction array. Finally, bacterial products produced by S. aureus biofilms will be identified using nuclear magnetic resonance and mass spectrometry analysis, and the identified products will be screened to determine if they are apoptosis-inducing factors. The goals of this investigation include identifying the molecular pathways mediated by S. aureus biofilms and the specific bacterial products that induce KC apoptosis. Indentifying clearly defined signaling pathways and apoptosis- inducing bacterial products may provide a molecular rational for future chronic wound therapies and diagnostics, which may have a significant impact on patient health.